(A) Lamina joint bending response to various amounts of 24-epiBL by the micro-drop method. A drop of ethanol (1 μL) containing 0, 10, 100 or 1000 ng of 24-epiBL, respectively, was spotted onto the top of the lamina of seedlings which were germinated for 2 days and grown for 3 days at 30°C. Images were obtained after 3 days of incubation. (B) Statistical data for lamina joint bending angle assay described in (A). The leaf angle assay was performed in two biological repeats, and each repeat with five plants measured for each concentration and each type of plants. Data are presented as mean ± SE. (C) Lamina joint bending response to 1 μM 24-epiBL by the excised leaf segment method. Seeds were germinated for 2 days and then grown in the dark for 8 days at 30°C. Segments comprising of a part of the second leaf blade, lamina joint, and leaf sheath were floated on distilled water for 24 h and then incubated in 1 μM 24-epiBL for 48 h in the dark. The experiments were performed in triplicate with similar results. (D) qRT-PCR analysis of the expression level of OsD2 , OsDWARF4 , OsGSK2 , and OsBZR1 genes in WT, OsOFP8 -OE, and OsOFP8 -RNAi plants. Two-week-old seedlings were used for analysis, which was performed in three biological repeats, each with three seedlings. Data represent mean ± SE, *, p < ; **, p < , t-test. OsActin 1 was used as a control. (E) qRT-PCR analysis of the expression level of OsOFP8 under the treatment of 1 μM 24-epiBL. Two-week-old seedlings were used for analysis with two biological repeats, each repeat having three seedlings for control and treatment, respectively. Data represent mean ± SE. (F) BR signaling induces OsOFP8 protein accumulation. Arabidopsis protoplast cells were transfected with OsOFP8-YFP and treated with 10 μM BRZ for 12 hours (h). After the treatment, BRZ was washed off, and then the cells were treated with 1 μM BL and harvested at the indicated time points, the protein was extracted from the harvested cells and used for western blotting, YFP antibody was used to detect OsOFP8 protein. CBB, Coomassie Brilliant Blue.
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Abstract Brassinosteroids (BRs) regulate plant development and stress response. Although much has been learned about their roles in plant development, the mechanisms by which BRs regulate plant stress tolerance remain unclear. Chilling is a major stress that adversely affects plant growth. Here, we report that BR positively regulates chilling tolerance in tomato. BR partial deficiency aggravated chilling‐induced oxidized protein accumulation, membrane lipid peroxidation and decrease of maximum quantum efficiency of photosystem II (Fv/Fm). By contrast, overexpression of BR biosynthesis gene Dwarf or treatment with 24‐epibrassinolide (EBR) attenuated chilling‐induced oxidative damages and resulted in an increase of Fv/Fm. BR increased transcripts of RESPIRATORY BURST OXIDASE HOMOLOG1 (RBOH1) and GLUTAREDOXIN (GRX) genes, and BR‐induced chilling tolerance was associated with an increase in the ratio of reduced/oxidized 2‐cysteine peroxiredoxin (2‐Cys Prx) and activation of antioxidant enzymes. However, RBOH1‐RNAi plants failed to respond to EBR as regard to the induction of GRX genes, activation of antioxidant capacity, and attenuation of chilling‐induced oxidative damages. Furthermore, silencing of GRXS12 and S14 compromised EBR‐induced increases in the ratio of reduced/oxidized 2‐Cys Prx and activities of antioxidant enzymes. Our study suggests that BR enhances chilling tolerance through a signaling cascade involving RBOH1, GRXs and 2‐Cys Prx in tomato.